Redox Changes in Perfusates Following Intracerebral Penetration of Microdialysis Probes

Microdialysis probe insertion into rat cerebral cortex significantly affects the levels of redox-active substances in brain extracellular fluid. Ascorbic acid levels are high immediately after probe insertion, decline rapidly, and then rise as the rat recovers from anesthesia 5–8 hours after surgery. Uric acid is at a low level for 5 hours and then rapidly increases in parallel with ascorbic acid. High ascorbic acid levels immediately after probe insertion are likely due to a shift from intracellular to extracellular fluids, whereas the delayed increase in uric acid may be due to increased enzymatic formation. After removal from the brain, hydrogen peroxide (H₂O₂) in microdialysis samples produces catalase-sensitive oxidative chemiluminescence. Microdialysis samples also produce high level catalase-resistant chemiluminescence associated with ascorbic acid levels after penetration injury. Although ascorbic acid is likely an antioxidant at concentrations estimated to be in brain extracellular fluid, it may have prooxidant effects when complexed with transition metals released into the neuronal microenvironment during traumatic brain injury.     

Analysis of transgene integration sites in transgenic pigs by fluorescence in situ hybridization

The production of pigs transgenic for human decay accelerating factor (hDAF) as potential donors for clinical organ xenotransplantation was reported several years ago. For this purpose it is required that high levels of hDAF are expressed at relevant sites in transplantable organs. Currently, homozygous lines have been produced as well as lines from crosses between heterozygous animals from different founder lines, termed jigsaw pigs. The purpose of the jigsaw crosses is to combine the desirable hDAF protein expression patterns found in different founder lines. Initial selection of the jigsaw pigs is based on the inheritance of the hDAF integration sites from both lines. Litters with potential homozygous transgenics and jigsaw transgenics were analysed by fluorescence in situ hybridization (FISH) and slot blot analysis. Results show that both slot blot analysis and FISH are suitable to distinguish between pigs that are heterozygous and homozygous for hDAF. However, FISH has the advantage of producing results more rapidly. For the identification of jigsaw pigs FISH analysis was required since slot blot analysis lacked the required accuracy. On basis of these results, FISH analysis was made part of the routine screening programme for hDAF transgenic pigs     

The Effects of Mechanical Stimulation on Some Electrical Properties of Axons

Rapid, short duration mechanical compression of lobster giant axons by a crystal-driven stylus produces a depolarization and an increase in membrane conductance which develop immediately with compression but take several seconds to recover. The conductance increase occurs even when the depolarization is prevented electrically. If sodium is removed from the external medium or if procaine is added to it, compression produces almost no depolarization. Small bundles of myelinated frog fibers are depolarized by rapid compression but recover very rapidly (milliseconds); "off" responses are occasionally seen. The results are discussed in terms of the mechanoelectric transducer behavior of an axon membrane.     

Enterohepatic circulation in man. A simple method for the determination of duodenal bile acids

A method has been developed for easy sampling of duodenal bile acids. For this purpose Entero-Test was used, an encapsulated nylon thread originally used to estimate enteral parasites. This capsule is swallowed by a fasting subject and one end of the thread is taped at a corner of the month. Four hours after swallowing the thread, it is withdrawn and bile acids are eluted with buffer. The solution is applied to a Sep-Pak C18 cartridge to extract bile acids, which are subsequently analyzed by capillary gas-liquid chromatography and liquid chromatography. In vitro analyses showed that there was no preferential binding to the thread of any bile acid and that binding was pH-independent. A high correlation (r = 0.98) was found between direct analyses of bile and analyses by Entero-Test after in vitro incubation. The values obtained by the Entero-Test were similar to those of duodenal bile simultaneously collected with the normal intubation technique (r = 0.99). Duodenal bile acid composition showed a daily variation. In 11 healthy volunteers the following bile acid composition of unstimulated duodenal juice was found (mean +/- SD; %): choleate 44 +/- 12 (glycine/taurine ratio 1.8), chenodeoxycholate: 29 +/- 6 (G/T ratio 2.3); deoxycholate: 25 +/- 11 (G/T ratio 5.7), lithocholate: 1, ursodeoxycholate: less than 1. The described technique turned out to be an easily applicable method for determination of duodenal bile acids in man. This enables longitudinal studies concerning the factors that determine the bile acid pool composition and its relevance to various diseases.     

DEVELOPMENTAL INSTABILITY AND HIGH MERISTIC COUNTS IN INTERSPECIFIC HYBRIDS OF SALMONID FISHES

Interspecific hybrids have been proposed to have reduced developmental stability in comparison to their parental species because the parental genomes have not undergone selection for the maintenance of developmental stability when they occur together. We present data from four interspecific hybrids of salmonid fishes that support this view. Natural hybrids of bull trout (Salvelinus confluentus) with brook trout (Salvelinus fontinalis) and laboratory hybrids of rainbow trout (Salmo gairdneri) with Yellowstone (Salmo clarki bouvieri), westslope (S. c. lewisi), and coastal (S. c. clarki) cutthroat trout all have higher levels of fluctuating asymmetry than either of their parental species raised in the same environment. Thus, the hybrids have reduced developmental stability. The hybrids do not have meristic counts intermediate to the counts of the parental species. The hybrids usually have counts as high as the species with the higher count for those characters that differ between the parental species and often have higher counts for those characters that do not differ between the parental species. We suggest that the tendency for interspecific hybrids to have high meristic counts may be related to differences between the species in the length and timing of the developmental periods during which the counts of the characters are determined.     

Impact of a glossy collard trap crop on diamondback moth adult movement, oviposition, and larval survival

One component of developing a systematic approach for deployment of trap crops is to understand how the trap crop modifies pest behavior. Glossy‐leafed collards, Brassica oleracea L. var. acephala (Brassicaceae), were evaluated as a potential trap crop for diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), because they are attractive to P. xylostella adults and are a poor host for P. xylostella larvae compared to cabbage, Brassica oleracea L. var. capitata. We used large field plots to measure the changes in adult, egg, and larval P. xylostella densities in cabbage when the trap crop was planted in the field. Furthermore, we planted the trap crop in dispersed and concentrated spatial arrangements to determine the impact of trap crop arrangement on the behavior of P. xylostella. In 2002, results showed that the presence of collards within a cabbage field reduced larval density on cabbage. In 2003, neither trap crop arrangement had a significant impact on P. xylostella larval density on cabbage. Adult moths aggregated in proximity to collards in 2002, but not in 2003. Egg and larval data in both years in all treatments showed that total oviposition was highest near a central release point, indicating that females lay many eggs before dispersing very far when suitable host plants are available. The mean direction of P. xylostella movement and oviposition from a central release point was not consistent or correlated to wind direction. Plant size of the trap crop in relation to the main crop and environmental factors may have been responsible for the inconsistent effectiveness of the trap crop.     

Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D₃₁-16:0 (palmitic) acid yielded D₃₁-18:0 (stearic) acid, D₂₉-18:1 (oleic and cis-vaccenic) acids, and D₂₉-heptadecene. These findings showed that loss of the carboxyl group of a C₁₈ monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C₂₁ hexaene, without detectable C₁₅-C₁₉ hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C₁₆ and C₁₈ fatty acids into alka(e)nes by a new, light-dependent pathway.Hydrocarbons derived from fatty acids (i.e. alkanes and alkenes) are ubiquitous in plant and insect outermost tissues where they often represent a major part of cuticular waxes and play an essential role in preventing water loss from the organisms to the dry terrestrial environment (Hadley, 1989; Kunst et al., 2005). In several insect species, select cuticular alkenes also act as sex pheromones (Wicker-Thomas and Chertemps, 2010). Occurrence of alkanes or alkenes has also been reported in various microorganisms (Ladygina et al., 2006; Wang and Lu, 2013). For example, synthesis of hydrocarbons is widespread in cyanobacteria (Coates et al., 2014), and it is thought that cyanobacterial alka(e)nes contribute significantly to the hydrocarbon cycle of the upper ocean (Lea-Smith et al., 2015).Alka(e)nes of various chain lengths are important targets for biotechnology because they are major components of gasoline (mainly C5-C9 hydrocarbons), jet fuels (C5-C16), and diesel fuels (C12-C20). The alkane biosynthetic pathway of plants has been partly elucidated (Lee and Suh, 2013; Bernard and Joubès, 2013), but the use of plant hydrocarbons as a renewable source of liquid fuels is hampered by predominance of constituents with high carbon numbers (>C25), which entails solid state at ambient temperature (Jetter and Kunst, 2008). Therefore, there is great interest in the microbial pathways of hydrocarbon synthesis producing shorter chain compounds (C15-C19). In cyanobacteria, hydrocarbons are produced by two distinct pathways. The first one comprises the sequential action of an acyl-ACP reductase transforming a C15-C19 fatty acyl-ACP into a fatty aldehyde and an aldehyde-deformylating oxygenase catalyzing the oxidative cleavage of the fatty aldehyde into alka(e)ne and formic acid (Schirmer et al., 2010; Li et al., 2012). The second pathway involves a type I polyketide synthase that elongates and decarboxylates fatty acids to form alkenes with a terminal double bond (Mendez-Perez et al., 2011). Additional pathways of alkene synthesis have been described in bacteria. In Micrococcus luteus, a three-gene cluster has been shown to control the head-to-head condensation of fatty acids to form very-long-chain alkenes with internal double bonds (Beller et al., 2010). Direct decarboxylation of a long-chain fatty acid into a terminal alkene has also been reported and is catalyzed by a cytochrome P450 in Jeotgalicoccus spp. (Rude et al., 2011) and by a nonheme iron oxidase in Pseudomonas (Rui et al., 2014).Among microbes, microalgae would be ideally suited to harness the synthesis of hydrocarbons from fatty acid precursors because they are photosynthetic organisms combining a high biomass productivity (León-Bañares et al., 2004; Beer et al., 2009; Malcata, 2011; Wijffels et al., 2013) and a strong capacity to synthesize and accumulate fatty acids (Hu et al., 2008; Harwood and Guschina, 2009; Liu and Benning 2013). However, studies on microalgal alka(e)ne synthesis are scarce. In some diatoms and other algal species, a very-long-chain alkene, a C21 hexaene, has been found (Lee et al., 1970; Lee and Loeblich, 1971). Other very-long-chain alkenes have been described in the slow-growing colonial Chlorophycea Botryococcus brauni, which excretes a variety of hydrophobic compounds including C27 n-alkadienes (Metzger and Largeau, 2005; Jin et al., 2016). A decarbonylase activity converting a fatty n-aldehyde substrate to a n-alkane has been shown in B. brauni (Dennis and Kolattukudy, 1992); however, the corresponding protein has not been identified so far. Presence of shorter chain alka(e)nes in some microalga species has been reported in the context of geochemical studies (Han et al., 1968; Gelpi et al., 1970; Tornabene et al., 1980; Afi et al., 1996) but the biology of these compounds has not been investigated further.Here, we show that alka(e)nes with C15 to C17 chains can be detected in several model microalgae and originate from fatty acid metabolism. In Chlamydomonas reinhardtii and Chlorella variabilis NC64A, 7-heptadecene is identified as the major hydrocarbon produced, and we demonstrate that its synthesis depends strictly on light and uses cis-vaccenic acid as a precursor. We also show the presence of C15 to C17 alka(e)nes in Nannochloropsis sp., a model microalga from the red lineage. Absence of homologs to known hydrocarbon synthesis genes in the genomes of Chlamydomonas, Chlorella, and Nannochloropsis indicates that a hitherto unknown type of alka(e)ne-producing pathway operates in these microalgae. The wide occurrence of microalgae in marine environments suggests that microalgal alka(e)nes contribute significantly to the ocean’s hydrocarbon cycle.     

Snake evolution in Melanesia: origin of the Hydrophiinae (Serpentes,Elapidae), and the evolutionary history of the enigmatic New Guinean elapid Toxicocalamus

The venomous snake subfamily Hydrophiinae includes more than 40 genera and approximately 200 species. Most members of this clade inhabit Australia, and have been well studied. But, because of poor taxon sampling of Melanesian taxa, basal evolutionary relationships have remained poorly resolved. The Melanesian genera Ogmodon, Loveridgelaps, and Salomonelaps have not been included in recent phylogenetic studies, and the New Guinean endemic, Toxicocalamus, has been poorly sampled and sometimes recovered as polyphyletic. We generated a multilocus phylogeny for the subfamily using three mitochondrial and four nuclear loci so as to investigate relationships among the basal hydrophiine genera and to determine the status of Toxicocalamus. We sequenced these loci for eight of the 12 described species within Toxicocalamus, representing the largest molecular data set for this genus. We found that a system of offshore island arcs in Melanesia was the centre of origin for terrestrial species of Hydrophiinae, and we recovered Toxicocalamus as monophyletic. Toxicocalamus demonstrates high genetic and morphological diversity, but some of the molecular diversity is not accompanied by diagnostic morphological change. We document at least five undescribed species that all key morphologically to Toxicocalamus loriae (Boulenger, 1898), rendering this species polyphyletic. Continued work on Toxicocalamus is needed to document the diversity of this genus, and is likely to result in the discovery of additional species. Our increased taxon sampling allowed us to better understand the evolution and biogeography of Hydrophiinae; however, several unsampled lineages remain, the later study of which may be used to test our biogeographic hypothesis.     

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, −10, and −9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH₂, has specificity constants of 6.3 (±0.3) × 10⁴ M⁻¹ s⁻¹ and 2.4 (±0.3) × 10³ M⁻¹ s⁻¹ for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH₂ and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH₂. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, −2, −3, −8, −9, −12, and −14. This substrate provides a unique tool in which to assess ADAM17, −10, and −9 activities.